11. Remove the nitrocellulose membrane containing the transferred proteins from the blotting cassette and examine the
transferred protein by staining with Ponceau S solution for
10 s and wash with deionized water for 3 times.
12. Block the membrane with 5 mL of 5% skim milk for 1 h at
room temperature and then incubate the membrane with primary antibody HB112 at a 1:500 dilution overnight.
13. Wash the membrane 3 times with TBS-T for 5 min each time.
Incubate the membrane with a HRP-conjugated goat antimouse IgG secondary antibody at a 1:5000 dilution for 1 h
at room temperature.
14. Wash the membrane 3 times with TBS-T for 5 min each time
before developing the signal by incubating the membrane with
an HRP substrate for 5 min in the dark (follow the manufacturer’s protocol).
15. Observe the signal using an Azure C400 signal detector or
other suitable detection equipment (Fig. 2).
3.5 Detection of Zika
VLP by Electron
Microscopy
1. Place a formvar-carbon film nickel grid onto a glass slide and
attach a small area of the grid to the slide using Scotch Brand
masking tape. Put the grid into a Quorum SC7620 Sputter
Coat machine and glow discharge at 18 mA for 120 s.
2. Directly drop 3 μL of purified Zika VLP in TNE buffer onto
the grid and incubate at room temperature for 1 min. Remove
the excess purified Zika VLP solution from the grid by applying
filter paper at the edge of the grid to absorb excess solution.
3. Directly drop 3 μL of 2% uranyl acetate onto the grid and
incubate at room temperature for 2 min (see Note 13). Remove
the excess uranyl acetate by filter paper and let the grid dry for
2 min at room temperature.
4. Wash the grid 1 time with deionized water followed by removing the excess water with filter paper and dry for 5 min at room
temperature.
5. Examine the Zika VLP under a transmission electron microscope (Fig. 2).
4 Notes
1. We purchased a commercially synthesized construct with
codon optimization for mammalian expression in vector
pUC57, as described in a previous publication [13]. pUC57
is a standard prokaryotic cloning vector and the insert requires
transfer to a mammalian expression vector (in this case the
pcDNA™ 3.1(+) plasmid vector). Expression of the insert in
200
Atichat Kuadkitkan et al.
transferred protein by staining with Ponceau S solution for
10 s and wash with deionized water for 3 times.
12. Block the membrane with 5 mL of 5% skim milk for 1 h at
room temperature and then incubate the membrane with primary antibody HB112 at a 1:500 dilution overnight.
13. Wash the membrane 3 times with TBS-T for 5 min each time.
Incubate the membrane with a HRP-conjugated goat antimouse IgG secondary antibody at a 1:5000 dilution for 1 h
at room temperature.
14. Wash the membrane 3 times with TBS-T for 5 min each time
before developing the signal by incubating the membrane with
an HRP substrate for 5 min in the dark (follow the manufacturer’s protocol).
15. Observe the signal using an Azure C400 signal detector or
other suitable detection equipment (Fig. 2).
3.5 Detection of Zika
VLP by Electron
Microscopy
1. Place a formvar-carbon film nickel grid onto a glass slide and
attach a small area of the grid to the slide using Scotch Brand
masking tape. Put the grid into a Quorum SC7620 Sputter
Coat machine and glow discharge at 18 mA for 120 s.
2. Directly drop 3 μL of purified Zika VLP in TNE buffer onto
the grid and incubate at room temperature for 1 min. Remove
the excess purified Zika VLP solution from the grid by applying
filter paper at the edge of the grid to absorb excess solution.
3. Directly drop 3 μL of 2% uranyl acetate onto the grid and
incubate at room temperature for 2 min (see Note 13). Remove
the excess uranyl acetate by filter paper and let the grid dry for
2 min at room temperature.
4. Wash the grid 1 time with deionized water followed by removing the excess water with filter paper and dry for 5 min at room
temperature.
5. Examine the Zika VLP under a transmission electron microscope (Fig. 2).
4 Notes
1. We purchased a commercially synthesized construct with
codon optimization for mammalian expression in vector
pUC57, as described in a previous publication [13]. pUC57
is a standard prokaryotic cloning vector and the insert requires
transfer to a mammalian expression vector (in this case the
pcDNA™ 3.1(+) plasmid vector). Expression of the insert in
200
Atichat Kuadkitkan et al.